Preventive cancer vaccine based on brother of regulator of imprinted sites molecule

ABSTRACT

Polynucleotides encoding a nonfunctional mutant form of the Brother of Regulator of Imprinted Sites (BORIS) molecule, nonfunctional mutated BORIS protein, polypeptide or peptide and modified protein forms of BORIS are described. These molecules are used as a therapeutic vaccine against cancer.

FIELD OF THE INVENTION

The present invention relates to compositions and methods used for thegeneration of a tumor vaccine.

BACKGROUND

Vertebrates possess the ability to mount an immune response as a defenseagainst pathogens from the environment as well as against aberrantcells, such as tumor cells, which develop internally. The immuneresponse is the result of complex interactions between a variety ofcells and factors, but generally comprises two main facets. One is acellular component, in which specialized cells directly attack anoffending agent (bearing an antigen) while the other is a humoralcomponent, in which antibody molecules bind specifically to the antigenand aid in its elimination. Acting in concert, the individual elementsare quite effective in limiting the initial onslaught of invadingpathogens and eliminating them from the host.

The primary cells involved in providing an immune response arelymphocytes, which generally comprise two principal classes. The firstof these, designated B cells or B lymphocytes, are typically generatedin bone marrow and are, among other duties, responsible for producingand secreting antibodies. B cell antibody products tend to reactdirectly with foreign antigens and neutralize them or activate othercomponents of the immune systems that then eliminate them. Inparticular, opsonizing antibodies bind to extracellular foreign agentsthereby rendering them susceptible to phagocytosis and subsequentintracellular killing. On the other hand, T cells or T lymphocytes,which generally develop or mature in the thymus, are responsible formediating the cellular immune response. These cells do not recognizewhole antigens but, instead, respond to short peptide fragments thereofbound to specialized proteins that appear on the surface of the surfaceof a target cell as well as an antigen presenting cell. Moreparticularly, it appears that proteins produced within the cell, ortaken up by the cell from extracellular milieu, are continually degradedto peptides by normal metabolic pathways. The resulting short fragmentsassociate with intracellular major histocompatibility complex (MHC)molecules and the MHC-peptide complexes are transported to the surfaceof the cell for recognition by T cells. Thus, the cellular immune systemis constantly monitoring a full spectrum of proteins produced oringested by the cells and is posed to eliminate any cells presentingforeign antigens or tumor antigens; i.e. virus infected cells or cancercells.

The structure of immunoglobulin G (IgG) is that of a tetrameric proteincomplex comprising two identical heavy (H) chains and two identicalimmunoglobulin light (L) chains. These chains are joined together bydisulfide bonds to form the Y-shaped antibody complex. In solutionhowever, the molecule takes on a more globular shape and readily bind toforeign antigens present in biological fluids. Amino acid sequenceanalysis of immunoglobulins has led to the definition of specificregions with various functional activities within the chains. Each lightchain and each heavy chain has a variable region (V_(L) and V_(H)respectively) defined within the first 110 amino terminal residues.Three dimensional pairing of the V_(L) and V_(H) regions constitute theantigen-recognition portion or “antigen combining site” (“ACS”) ofimmunoglobulin molecule. Because of the tetrameric nature ofimmunoglobulins, there are two identical antigen combining sites permolecule. The variable domains of these chains are highly heterogeneousin sequence and provide the diversity for antigen combining sites to behighly specific for a large variety of antigenic structures. Theheterogeneity of the variable domains is not evenly distributedthroughout the variable regions, but is located in three segments,called complementarity determining regions (“CDRs”) designated CDR 1,CDR 2 and CDR 3. For further information regarding these structures seeWatson et al., 1987, Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings Publishing Co., Inc. Menlo Park, Calif. incorporatedherein by reference.

Each of the heavy chains also includes a constant region defining aparticular isotype and assigns the immunoglobulin to one of theimmunoglobulin classes and subclasses. The constant region containsunits called domains (i.e. C_(H1), C_(H2), etc.) that do not varysignificantly among antibodies of a single class. The constant regiondoes not participate in antigen binding, but can be associated with anumber of biological activities known as “effector functions”, such asbinding to Fc receptors on cell surfaces as well as binding tocomplement proteins. Antigen presenting cells such as dendritic cellsand macrophages are, among other features, generally distinguished bythe presence of an Fc receptor. Consequently, if an antibody is bound toa pathogen, it can then link to a phagocyte via the Fc portion. Thisallows the pathogen to be ingested and destroyed by the phagocyte, aprocess known as opsonization. Moreover, as will be discussed in moredetail below, various pathogenic antigens may be processed and displayedby the APC to further stimulate an immune response.

Unlike the heavy chains, the light chains have a single constant domain(C_(L)). A light chain pairs with a heavy chain through a disulfide bondwhich attaches heavy constant region C_(H1) to C_(L). In addition, theheavy chains have a hinge region separating constant regions C_(H1) andC_(H2) from the remainder of the molecule. It is this hinge region thatis largely responsible for the flexibility of the tetramer. The twoheavy chains of the molecule pair together through disulfide bonds atthe junction between the hinge region and C_(H2).

In order to provide such an extensive repertoire, immunoglobulin geneshave evolved so as permit the production of vast numbers of differentimmunoglobulin proteins from a finite number of genes i.e. inherentpolymorphism. Due to inherent polymorphism, mammals are able to produceantibodies to a seemingly infinite variety of antigens. For a review ofimmunoglobulin genetics and protein structure see Lewin, “Genes III”,John Wiley and Sons, N.Y. (1987) and Benjamini and Leskowitz, 1988,Immunology, Alan R. Liss, Inc., New York, which is incorporated hereinby reference.

In the past few years antibodies have become extremely important indiagnostic and therapeutic applications due to their diversity andspecificity. Increasingly, molecular biology techniques have been usedto expand the variety and availability of antibodies for scientificapplications. For instance, a single antibody producing B cell can beimmortalized by fusion with a tumor cell and expanded to provide an invitro source of antibodies of a single specificity known as a“monoclonal antibody” (mAb). Such an immortal B cell line is termed a“hybridoma.”

Until recently, the source of most mAb has been murine (mouse)hybridomas cultured in vitro. That is, a mouse was typically injectedwith a selected antigen or immunogen. Subsequently, the animal wassacrificed and cells removed from its spleen were fused with immortalmyeloma cells. Although they have been used extensively in diagnosticprocedures, murine mAb are not well suited for therapeutic applicationsin most mammals including humans. In part, this is due to the fact thatmurine antibodies are recognized as foreign by other mammalian speciesand elicit an immune response that may itself cause illness.

To overcome at least some of the problems of immune responses generatedby foreign mAb and the lack of suitable human mAb, genetic engineeringhas been used to construct humanized chimeric immunoglobulin moleculeswhich contain the antigen binding complementarity determining regions ofthe murine antibodies but in which the remainder of the molecule iscomposed of human antibody sequences which are not recognized asforeign. Such antibodies have been used to treat tumors as the mousevariable region recognizes the tumor antigen and the humanized portionof the molecule is able to mediate an immune response without beingrapidly eliminated by the body. See, for example, Jones et al., Nature,321:522-525 (1986), which is incorporated herein by reference.

Other uses of such antibodies are detailed in PCT Publication No. WO94/14847, which is also incorporated herein by reference. In these casesepitopes of foreign antigens such as viral or bacterial epitopes aregrafted onto the hypervariable region of an immunoglobulin to induce aresponse. That is, the engineered antibodies are used as a vaccine toprovoke an immune response and confer long-term immunogenic memorythereby allowing the subject to fight off subsequent infections.

These and more traditional vaccines are effective in that they stimulateboth prongs of the immune system. Despite the intricacies associatedwith the humoral component of the immune response, it would not, in andof itself, be capable of effectively protecting an animal from themyriad pathogenic assaults to which it is subject each day. Rather, itis only the presence of a highly evolved cellular response that allowshigher organisms to survive and proliferate.

As indicated above, T lymphocytes or T cells, which arise fromprecursors in the bone marrow, are central players in the immuneresponse against invading viruses and other microbes. The progenitorstem cells migrate to the thymus where, as so-called thymocytes, theybecome specialized. In particular, they begin to display the receptormolecules that later enable mature T cells to detect infection To bebeneficial, T cells must be able to attach through their receptors toantigens (protein markers signaling an invader's presence). At the sametime, they should be blind to substances made by the body asself-reactive T cells can destroy normal tissues. Typically, only thosethymocytes that make useful receptors will mature fully and enter thebloodstream to patrol the body. Others that would be ineffectual orwould attack the body's own tissue are, in healthy individuals,eliminated through apoptosis prior to leaving the thymus.

Mature T cells that finally enter the circulation, either as cytolytic Tlymphocytes or T helper cells, remain at rest unless they encounterantigens that their receptors can recognize. Upon encountering thespecific antigens for which the lymphocytes have affinity, theyproliferate and perform effector functions, the result of which iselimination of the foreign antigens.

T cells have been classified into several subpopulations based on thedifferent tasks they perform. These subpopulations include helper Tcells (T_(h)), which are required for promoting or enhancing T and Bcell responses; cytotoxic (or cytolytic) T lymphocytes (CTL), whichdirectly kill their target cells by cell lysis; and suppressor orregulatory T cells (T_(s) or Tr) which down-regulate the immuneresponse. In every case T cells recognize antigens, but only whenpresented on the surface of a cell by a specialized protein complexattached to the surface of antigen presenting cells. More particularly,T cells use a specific receptor, termed the T cell antigen receptor(TCR), which is a transmembrane protein complex capable of recognizingan antigen in association with the group of proteins collectively termedthe major histocompatibility complex (MHC). Thousands of identical TCR'sare expressed on each cell. The TCR is related, both in function andstructure, to the surface antibody (non-secreted) which B cells use astheir antigen receptors. Further, different subpopulations of T cellsalso express a variety of cell surface proteins, some of which aretermed “marker proteins” because they are characteristic of particularsubpopulations. For example, most T_(h) cells express the cell surfaceCD4 protein, whereas most CTL cells express the cell surface CD8 proteinand Tr cells expressed CD25 and CD4 molecules. These surface proteinsare important in the initiation and maintenance of immune responses thatdepend on the recognition of, and interactions between, particularproteins or protein complexes on the surface of APCs.

For some time it has been known that the major histocompatibilitycomplex or MHC actually comprises a series of glycosylated proteinscomprising distinct quaternary structures. Generally, the structures areof two types: class I MHC which displays peptides from proteins madeinside the cell (such as self-proteins or proteins produced subsequentto viral replication), and class II MHC, which generally displayspeptides from proteins that have entered the cell from the outside(soluble antigens such as bacterial toxins). Recognition of variousantigens is assured by inherited polymorphism that continuously providesa diverse pool of MHC molecules capable of binding any pathogenicpeptides that may arise. Essentially, all nucleated cells produce andexpress class I MHC, which may exhibit naturally occurring peptides,tumor associated peptides or peptides produced by a viral invader.Conversely, some other nucleated cells and among them specializedlymphoid cells, those generally known as antigen presenting cells,produce and express class II MHC proteins. Regardless of the cell type,both classes of MHC carry peptides to the cell surface and present themto resting T lymphocytes. Ordinarily, T_(h) cells recognize class IIMHC-antigen complexes while CTL's tend to recognize class I MHC-antigencomplexes, although cross-presentation of antigens also occurred

When a resting T cell bearing the appropriate TCR encounters the APCdisplaying the peptide on its surface, the TCR binds to the peptide-MHCcomplex. More particularly, hundreds of TCR's bind to numerouspeptide-MHC complexes. When enough TCRs are contacted the cumulativeeffect activates the T cell. Receptors on T cells that are responsiblefor the specific recognition of, and response to, the MHC-antigencomplex are composed of a complex of several integral plasma membraneproteins. As with the MHC complex previously discussed, a diverse poolof TCR's is assured by inherent polymorphism leading to somaticrearrangement. It should be emphasized that, while the pool of TCR's maybe diverse, each individual T cell only expresses a single specific TCR.However, each T cell typically exhibits thousands of copies of thisreceptor, specific for only one peptide, on the surface of each cell. Inaddition, several other types of membrane associated proteins areinvolved with T cell binding and activation.

Activation of the T cell entails the generation of a series of chemicalsignals (primarily cytokines) that result in the cell taking directaction or stimulating other cells of the immune system to act. In thecase of class I MHC-antigen activation, CTL's proliferate and act todestroy infected cells presenting the same antigen. Killing an infectedcell deprives a virus of life support and makes it accessible toantibodies, which finally eliminate it. In contrast, activation of T_(h)cells by class II MHC-antigen complexes does not destroy the antigenpresenting cell (which is part of the host's defense system) but ratherstimulates the T_(h) cell to proliferate and generate signals (againprimarily cytokines) that affect various cells. Among otherconsequences, the signaling leads to B cell stimulation, macrophageactivation, CTL differentiation and promotion of inflammation. Thisconcerted response is relatively specific and is directed to foreignelements bearing the peptide presented by the class II MHC system.

Constant surveillance of epitopes throughout those structures in thebody accessible to the immune system provides a very effective means forrecognizing and maintaining “self” and destroying epitopes and theircarriers that invade the body or arise pathologically. When operatingproperly the immune response is surprisingly effective at eliminatingmicroscopic pathogens and neoplastic (tumor) cells that are believed toarise continuously in the body and for the most part are eliminated bythe immune system before becoming detectable. Certain regions of thebody, such as the brain, eye, and testis, are protected from immunesurveillance, these sites are referred to as immune privileged. Ingeneral, the complicated mechanisms for self-recognition are veryefficient and allow a strong response to be directed exclusively atforeign antigens. Unfortunately, the immune system occasionallymalfunctions and turns against the cells of the host provoking anautoimmune response. Typically, autoimmunity is held to occur when theantigen receptors on immune cells recognize specific antigens on healthycells and cause the cells bearing those particular substances to die. Inmany cases, autoimmune reactions are self-limited in that they disappearwhen the antigens that set them off are cleared away. However, in someinstances the autoreactive lymphocytes survive longer than they shouldand continue to induce apoptosis or otherwise eliminate normal cells.

Current data indicates that immune protection against all cancersrequires the generation of a potent cellular immune responses against aunique tumor antigen expressed by the malignant cell. As a consequence,successful immune protection first requires a unique antigen expressedin the tumor cells (tumor-specific antigen) and second, induction of apotent T cell immune response targeted to the tumor antigen.

Several tumor-associated antigens are currently known, and have beenused in pre-clinical and clinical studies for generating vaccines. Forexample, PSMA, PAP and PSA are antigens expressed in prostate tumorcells. Her2/neu and MUC1 are antigens expressed by breast cancer cellsand other carcinomas, including carcinomas of the lung, ovary, colon,and pancreas. MAGEs and MART-1 are melanoma tumor cell-associatedantigens, and CEA is an antigen associated with pancreas or colorectalcancer. Other tissue and/or tumor specific antigens also have beendescribed. However, while all of these antigens are expressed in tumorcells in the normal or aberrant forms, they are also expressed in avariety of normal cells, and thus cannot be used for prophylacticvaccination. In other words, these tumor-associated antigens are stillrecognized by immune cells as self-molecules and so no true activationof the immune system occurs. This presents at least two obstacles fortargeting these tumor-associated molecules as the basis for a vaccine.The first obstacle is the unresponsiveness (tolerance) of the immunesystem to self-molecules, which restricts its ability to generate potentcellular immune responses. The second is that any potent cellular immuneresponse generated should not be directed toward normal cells thatexpress the target antigen. This is the reason that all thetumor-associated antigens discussed above are suggested for use only astargets for therapeutic vaccinations.

A new protein has been recently described that is able to overcome theproblems associated with the known tumor-associated antigens. Brother ofRegulator of Imprinted Sites (BORIS) was first described as aDNA-binding protein found in testis. This protein shares 11 zinc-finger(ZF) domains with CCCTC-binding factor (CTCF) that is a multivalent11-zinc finger nuclear factor. CTCF is a conserved, ubiquitous andhighly versatile factor involved in various aspects of gene regulationand which forms methylation-sensitive insulators that regulate Xchromosome inactivation and expression of imprinted genes. BORIS differsfrom CTCF, however, at the N and C termini and is expressed in amutually exclusive manner with CTCF during male germ cell development.BORIS expression is restricted to the testis and then only within aselect cell subpopulation of spermatocytes that are involved with there-setting of methylation marks during male germ cell development. Thistestis cell subpopulation is also the only normal cell type known thatdoes not express CTCF. Because inhibition of CTCF expression in culturedcells leads to apoptosis, it is reasonable to assume that BORIS isactivated to maintain some of the vital CTCF functions in testis cells(Loukinov et al. (2002) Proc. Natl. Acad. Sci. 99(10):6806-6811; whichis incorporated herein by reference).

More recently, it was demonstrated that while CTCF overexpression alsoblocks cell proliferation, expression of BORIS in normallyBORIS-negative cells promotes cell growth that can lead totransformation (Klenova et al. (2002) Cancer Biol. 12:399-414; which isincorporated herein by reference). Human BORIS maps to the 20q13 region,which is well known for frequent gains and/or amplifications observed inmany of the same types of tumors that also often show loss ofheterozygosity (LOH) at the paralogous locus on 16q22 where CTCFresides. These regions are associated with “hot-spots” associated withbreast, prostate, ovarian, gastric, liver, endometrial, glioma, colonand esophageal cancer as well as Wilms tumors. Importantly, abnormalactivation of BORIS expression appears to be found in a significantproportion of a wide variety of neoplasms. Using Northern blots orRT-PCR, Klenova et al. (2002) analyzed BORIS mRNA levels in over 200cancer cell lines representing most of the major forms of human tumorsand detected transcripts in more than one half of the cell lines tested.Subsequent analysis of primary cancers, for breast cancer samples,confirmed the results obtained with the cell lines.

SUMMARY OF THE INVENTION

The present invention is directed to nonfunctional mutantpolynucleotides encoding the Brother of Regulator of Imprinted Sites(BORIS) tumor antigen and the use of such polynucleotides for preventivevaccination and immunotherapy of primary or metastatic cancer. Thepolynucleotide may be either DNA or RNA. In one preferred embodiment,the tumor antigen is a non-functional mutated form of the BORIS moleculelacking DNA binding capability. In another preferred embodiment at leastone zinc finger (ZF) domain is nonfunctional due to mutation or deletionand the function of BORIS is eliminated. In another preferred embodimentany combination of the zinc finger domains are mutated or deleted andfunction of the BORIS protein, polypeptide or peptide is eliminated. Inyet another preferred embodiment all of the ZF binding sites aredeleted. In still another preferred embodiment the polynucleotideencoding the mutated form of BORIS is fused to a molecular adjuvant. Instill another preferred embodiment the polynucleotide encoding thenonfunctional mutated form of BORIS is mixed with at least one otherpolynucleotide encoding a molecular adjuvant. Any molecular adjuvantthat increases cellular immune response can be used. Cytokines,chemokines and co-stimulatory molecules are particularly preferred.Particularly preferred chemokines, cytokines and co-stimulatorymolecules are beta-defensin2, I112, IL18, MIPα3, IFNγ and CD80/86.

The present invention is also directed to a vector comprising apolynucleotide encoding a nonfunctional mutated form of BORIS. In apreferred embodiment the vector directs expression in a bacterial,mammalian, yeast cell or viral system.

The present invention is further directed to a nonfunctional modified(mutant) form of a BORIS protein, polypeptide or peptide. Thenonfunctional mutant can be made using any method that introducesdeletions, substitutions or additions in the sequence that result in anon-functional protein. In a preferred embodiment the mutant BORISprotein, polypeptide or peptide lacks DNA binding ability. In anotherpreferred embodiment the mutant BORIS protein, polypeptide or peptide ismixed with conventional adjuvant. In yet another preferred embodimentthe nonfunctional mutant BORIS protein, polypeptide, or peptide isattached to a. pharmaceutically acceptable carrier (backbone). In stillanother preferred embodiment the nonfunctional mutant BORIS protein,polypeptide or peptide is attached to a peptide that modifies BORIS andretains it antigenic property. In yet another preferred embodiment thenonfunctional mutant BORIS protein, polypeptide or peptide is attachedto a protein transducing domain (PTD).

The present invention is also directed to dendritic cells expressing anonfunctional mutant BORIS molecule. In a preferred embodiment thedendritic cells are transfected with DNA encoding a mutant BORISmolecule. In yet another preferred embodiment the dendritic cells areinfected with a viral vector that encodes a nonfunctional mutant BORISmolecule. In yet another preferred embodiment the dendritic cells areloaded with nonfunctional mutant BORIS protein, polypeptide, peptide orany nonfunctional modified protein form of BORIS.

The present invention encompasses cellular immune responses generatedagainst a nonfunctional mutant form of the BORIS protein, polypeptide,peptide or any nonfunctional modified protein form of BORIS. The presentinvention encompasses antibodies raised against a nonfunctional mutantform of the BORIS protein, polypeptide, peptide or any modified proteinform of BORIS.

The present invention also encompasses a cancer preventive ortherapeutic vaccine comprising a polynucleotide encoding a nonfunctionalmutant form of BORIS, a nonfunctional mutant BORIS protein, polypeptideor peptide or dendritic cells expressing a nonfunctional mutant BORISmolecule.

The present invention is also directed towards a method of treatingcancer comprising administering to a patient (prophylactic vaccine) inneed thereof an effective amount of a polynucleotide encoding anonfunctional mutant form of BORIS, a nonfunctional mutant BORISprotein, polypeptide or peptide, or dendritic cells expressing orcontaining a nonfunctional mutant BORIS molecule. Administration can bevia an intramuscular, subcutaneous, intradermal, intravenous, nasal,rectal, vaginal or peritoneal route. The cancer can be a primary ormetastatic cancer. The patient can have multiple different types ofcancer. In a preferred embodiment the cancer is breast, prostate,ovarian, gastric, liver, endometrial, glioma, colon, or esophagealcancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a, b, and c present the results of vaccination of mice (n=10)with pBORIS (DNA immunization) pIL12/IL18 (molecular adjuvant). Thisresulted in protection of mice from challenge with 10⁴ 4T1 tumor cellsnaturally expressing mouse BORIS. FIG. 1 a shows survival rate on the Yaxis and Days after tumor challenge on the X axis for pBORIS/pIL12/IL18,pIL12/IL18 and vector only. FIG. 1 b show the relationship between tumorvolume and days after tumor challenge for pBORIS/pIL12/IL18, pIL12/IL18and vector only. FIG. 1 c shows the significant difference in tumorvolume at day 21 between groups immunized with pBORIS/pIL12/IL18 vs.pIL12/IL18 and vector only (*P<0.001).

FIG. 2 a and b show the relationship between percent survival and daysafter tumor challenge (a) and tumor volume and days after challenge formice vaccinated with pBORIS (DNA immunizations) followed by Ad5-BORIS(viral like particles) and challenged with 10⁴ 4T1 cells. Datademonstrated a full protection against the tumor challenge after alleast 33 days of challenge.

FIG. 3 a, b, c present the results of gene-gun immunization of mice withpBORIS plus pIFNγ or pIL12/IL18 followed by challenge with 10⁵ 4T1 tumorcells. FIG. 3 a shows a prolonged time of tumor growth to the volume of2 cm³ and FIG. 3 b shows a lower the tumor growth rate. FIG. 3 c showssignificant differences in tumor volume at day 14 between groupspBORIS/pIFNγ vs. vector (p<0.05), pBORIS/pIL12/IL18 vs. pIL12/IL18(P<0.05) and pBORIS/pIL12/IL18 vs. vector (P<0.01).

FIG. 4 a, b, and c show the results of mice vaccinated with pBORIS (DNAimmunization) followed by injection of Ad5-BORIS (viral like particles).FIG. 4 a shows a significantly prolonged survival of the vaccinated micewhile FIG. 4 b shows that these mice had a lower the tumor growth rateand prolonged the time of tumor growth to the volume of 2 cm³ afterchallenge of mice with 10⁵ 4T1 tumor cells. FIG. 4 c shows a significantdifference in tumor volume at day 15 between groups Ad5-BORIS vs. Ad5(P<0.001).

FIG. 5 shows the best-fit alignment of the human and mouse BORISpolypeptides produced by the GCG-package of programs with zero-penaltyfor the gap extension with conserved zinc finger regions highlighted andindicated as ZF-11.

DETAILED DESCRIPTION OF THE INVENTION

While the present invention may be embodied in many different forms,specific illustrative embodiments are disclosed herein that exemplifythe principles of the invention. It should be emphasized that thepresent invention is not limited to the specific embodimentsillustrated.

The present invention involves the use of an antigen expressed only inimmune-privileged testis cells and appearing in many transformed tumorcells to prevent a tolerating effect, which may induce other tumorantigens. The invention also involves the introduction of specificchanges in the DNA encoding the antigen to eliminate side effects andautoimmune reactions. In this context the following definitions apply.

The terms “tumor,” “cancer,” “neoplasm,” “neoplasia” and theiretymological relatives are used interchangeably in the context of thisapplication to refer generally to dysproliferative diseases and theattendant affected cells or cell masses. Preferably, thedysproliferative cells referred to herein express an immune-privilegedantigen.

Cytotoxic T lymphocytes (CTLs) are effector T cells, usually CD8+ thatcan mediate the lysis of target cells bearing antigenic peptidesassociated with a MHC molecule. Other cytotoxic cells includegamma/delta and CD4+ NK 1.1+ cells.

Immune privilege and immune-privileged antigen refer to the isolation ofcertain sites and antigens within the body from the immune system andthus relate to antigens to which an immune response is not normallydeveloped. Immune-privileged antigens expressed ectopically (i.e.,outside of their normally immune-privileged sites) may result inautoimmunity or tumor immunity. Immune-privileged antigens are expressedby some tumors resulting in an immune response to both the tumor and tonon-tumor sites expressing the same immune-privileged antigens.

Antigen presenting cells (APCs) are cells including dendritic cells,macrophages, and B cells, that can process and present antigenicpeptides in association with class I or class II MHC molecules anddeliver a co-stimulatory signal necessary for T cell activation.

A “zinc finger domain” refers to a small independently folded domainthat requires coordination of one or more zinc ions to stabilize itsstructure. Fingers bind to three base pair subsites and specificcontacts are mediated by amino acids in positions-1, 2, 3 and 6 relativeto the start of the alpha helix.

A “nonfunctional mutant form of BORIS” refers to a BORIS protein,polypeptide or peptide that lacks function. “Lack of function” isintended to mean failing to perform any one of the critical activitiesof the wildtype BORIS molecule such as DNA binding, re-establishment ofthe paternal DNA-methylation pattern, etc.

“Nonfunctional mutant” refers to changes at the DNA or amino acid levelthat destroy the wildtype activity of the resulting protein. Suchchanges can be amino acid substitutions, deletions or additions in areasof the molecule that act as catalytic sites and/or participate inbinding DNA or protein. Examples of changes that are able to destroyactivity are deletions or substitutions of critical amino acidsparticipating in a catalytic or binding interaction, additions of aminoacids that alter the required three dimensional structure of the siteinvolved in catalytic and/or binding interactions, or additions ordeletions of nucleotides that cause frame shifts, thus destroying therequired three dimensional structure. Mutations can be produced usingcommon molecular techniques such as PCR, use of oligonucleotides, etc.(for example see Sambrook, Maniatis and Fritsch). Naturally occurringmutations can also be isolated from cell populations (for example seeSambrook, Maniatis and Fritsch).

A “peptide” refers to a molecule containing at least 2 amino acidsjoined by a peptide bond. A “polypeptide” refers to a moleculecontaining at least 10 amino acids joined by peptide bonds and a“protein” refers to a molecule containing at least 20 amino acids.

A “polynucleotide encoding a nonfunctional mutant form of BORIS” refersto any polynucleotide having at least 50%, 60% or 70% sequence identitywith the human (SEQ ID NO: 1) or mouse (SEQ ID NO: 3) BORISpolynucleotide, more likely 75%, 80%, 90%, 95% or 96%, 97%, 98% or 99%sequence identity with the human (SEQ ID NO: 1) or mouse (SEQ ID NO: 3)BORIS polynucleotide.

A “nonfunctional mutant BORIS peptide, polypeptide or protein” refers toa BORIS molecule that fails to perform any one of the criticalactivities of the wildtype BORIS molecule such as DNA binding,re-establishment of the paternal DNA-methylation pattern, etc. The“nonfunctional mutant BORIS peptide, polypeptide or protein” has atleast 50%, 60% or 70% sequence identity with the human (SEQ ID NO: 1) ormouse (SEQ ID NO: 2) BORIS peptide, polypeptide or protein, more likely75%, 80%, 90%, 95% or 96%, 97%, 98% or 99% sequence identity with thehuman (SEQ ID NO: 2) or mouse (SEQ ID NO: 4) BORIS peptide, polypeptideor protein.

A nonfunctional mutated BORIS molecule is recognized as a non-selfantigen expressed only in transformed tumor cells and is used as anantigen to overcome the limitations of the prior art. The mutant form ofBORIS is used as an ideal non-toxic vaccine, because it should not haveany undesirable side effects caused by its DNA-binding activity and/ornative function. In other words, the mutant BORIS used for vaccinationhas no functional activity and is present only as an immunogen(antigen). Unlike other tumor-specific antigens, BORIS is not expressedin the normal tissues in women. Furthermore, even though BORIS isexpressed during the pubertal development of the normal testis in men,introduction and/or expression of a nonfunctional mutant BORIS shouldnot be harmful, because the testis is an immune-privileged tissue(inaccessible for immune cells). In other words, the anti-BORIS immuneresponse generated after immunization is not dangerous for normal cellsand a BORIS vaccine does not induce autoimmunity. In addition,generation of a potent immune response is guaranteed because BORIS,unlike other tumor-specific antigens, is recognized as a foreignantigen. BORIS specific T cells are not deleted in thymus and recognizemutant BORIS as a non-self antigen and generate an immune response.

In one embodiment cDNA encoding mouse (mBORIS) BORIS is generated byRT-PCR on mRNA isolated from mouse or human testis. The DNA bindingdomain of the molecule is deleted and substituted with a small spacerknown to work well in creating single chain Fv domain antibodies. Thecorrect sequence is confirmed by automated nucleotide sequence analysis.The resulting molecule lacks the 11 ZF domains and consists of the Nterminal region of mBORIS (amino acids 1-258) linked to the C terminalregion (amino acids 573-636) through an 18-amino acid spacer.

The mutated cDNA is cloned into a pORF vector under control of thehEF1-HTLV promoter, however other expression vectors can be used. Here,the mutated cDNA is operably linked to a promoter and/or regulatorymolecules that are capable of causing expression in the host cell. Viralvectors can be used including α-virus DNA or RNA vectors, adenovirusesand retroviruses (see Vasilevko, V. et al. (2003) Clin. Exp. Metastas.20:489-98.; Leitner, W. W. et al. (2003) Nat Med 9:33-39; Ribas, A etal. (2002) Curr.Gene Ther 2:57-78).

In addition to the above, the invention encompasses using viral likeparticles encoding nonfunctional mutant BORIS molecules such as thosefrom adenovirus, human hepatitis B, human hepatitis C, vaccinia virus,polyoviurs, etc. Recombinant viral proteins from different viruses havethe useful property of self-assembling into virus-like particles (VLPs).These particles contain no viral nucleic acids and are thereforenon-replicative, non-infectious and retain conformationally correctantigenic epitopes. VLP production has been shown in many experimentalsystems, such as mammalian cells, baculovirus-infected insect cells,yeasts, E. coli, cell free systems and transgenic plants. Importantly,vaccination with VLPs generates production of not only humoral but alsocellular immune responses. VLPs infect professional APCs andsubsequently induce protective cellular immune responses, includingCD4⁺Th1 (type of CD4⁺T cells that helps CD8⁺T cells) and CD8⁺ CTLresponses. Thus, VLPs have clearly revealed an exceptional capacity toactivate cellular immune responses (T cell reponses). The potential useof VLPs as prophylactic vaccines is currently being assessed in a numberof different clinical trials. Results from these trials have beenencouraging with excellent tolerability and high immunogenicity reportedin each trial. Generation of a VLPs vaccine composed of truncated BORISantigen will promote the induction of strong cellular immune responsesagainst cancer cells expressing this tumor associated antigen. HepatitisB virus (HBV) core antigen (HBcAg) and VSV are examples of suitableVLPs.

To generate a more robust cellular immune response, the truncated ormutated mBORIS is fused with molecular adjuvants such as B7costimulatory molecules, beta-defensin 2/3, MIP3α, IFNγ, cytokines,chemokines, etc. prior to cloning into the vector. Other suitablemolecular adjuvants are listed in the Table below XCL1 (Lymphotactin α,SCM-1α, IL-1 α, IL-1β ATAC) XCL2 (Lymphotactin β, SCM-1β, IL-2 ATAC)CCL1 (I-309, TCA3) IL-3 CCL2 (MCP-1, MCAF, JE) IL-4 CCL3 (MIP-1, αMIP-1αS, IL-5 LD78α) LD78β (MIP-1αP) IL-6 LD78γ IL-7 CCL4 (MIP-1β) IL-9CCL5 (RANTES) IL-10 CCL7 (MCP-3) IL-11 CCL8 (MCP-2) IL-12 CCL9 (MIP-1γ)IL13 CCL10 (CCF18) IL14 CCL11 (Eotaxin) IL-15 CCL12 (MCP-5) IL-16 CCL13(MCP-4, CKβ10) IL-17 CCL15 (HCC-2, Lkn-1, MIP-5, CC-2, IL-18 NCC-3,MIP-1δ) CCL16 (NCC-4, LEC, HCC-4, LMC, Mtn-1, IL-21 LCC-1, CKβ12) CCL17(TARC) IL-23 CCL18 (DC-CK1, PARC, MIP-4, AMAC-1, TNFα CKβ7) CCL19(exodus-3, ELC, MIP-3β, TNFβ CKβ11) CCL20 (exodus-1, MIP-3α, LARC, IFNαST38) CCL21 (exodus-2, SLC, 6-Ckine, TCA4, IFNβ CKβ9) CCL22 (MDC,ABCD-1, DC/B-CK) IFNγ CCL23 (MIP-3, MPIF-1, CKβ8-1) M-CSF CCL24 (MPIF-2,CKβ6, eotaxin-2) G-CSF CCL25 (TECK, Ckβ15) GM-CSF CCL26 (Eotaxin-3,MIP-4α) MIF CCL27 (ALP, Skinkine, ILC, ESkine, CD46 (MCP) CTAK) CXCL8(IL-8) CD27 (T14, S152) CXCL9 (mig) CD54 (ICAM-1) CXCL10 (γIP-10, crg-2)CD80 (B7-1, BB1) CXCL11 (H174, β-R1, I-TAC, IP-9) CD86 (B7-2, B70)CXCL12 (SDF-1α, SDF-1β, PBSF) CD134 (FLT3, STK-1) CXCL13 (BLC, BCA-1)CDw137 (4-1BB) CXCL14 (BRAK, bolekine) CDw150 (SLAM, IPO3) CX₃CL1(Fractalkine, neurotactin) CD153 (CD30L) Defensin (DFα, DFβ) CD161(NKR-P1A)

Alternatively, conventional adjuvants can be used such as Tween 80, 5%ethanol and Bupivacaine for DNA immunization. Other examples ofconventional adjuvants include mineral salts (such as aluminiumhydroxide and aluminium phosphate gels), oil emulsions and surfactantbased formulations such as MF59, QS21, AS08 [SBAS2] (oil-in-wateremulsion+MPL+QS21), Montanide ISA-51 and ISA-720, particulate adjuvantssuch as virosomes, AS04 ([SBAS4] Al salt with MPL), ISCOMS, polylactideco-glycolide (PLG), microbial derivatives (natural and synthetic)including monophosphoryl lipid A (MPL), Detox (MPL+M.Phlei cell wallskeleton), AGP[RC-529], DC_Chol, OM-174 (lipid A derivative), CpGmotifs, modified LT and CT (genetically modified bacterial toxins),endogenous immunomodulators such as GM-CSF, IL-12, Immudaptin, as wellas all other chemokines, cytokines and costimulatory molecules listed inthe table above and inert vehicles, such as gold particles.

Adjuvants can be either mixed with the polypeptide encoding anonfunctional mutant form of the Brother of Regulator of Imprinted Sites(BORIS) protein, polypeptide or peptide, a nonfunctional mutant BORISprotein, polypeptide or peptide and a dendritic cell expressing anonfunctional mutant BORIS peptide, polypeptide or protein

Additional peptide molecules can be included in the nonfunctional mutantBORIS constructs to enhance/promote presentation of the nonfunctionalmutant BORIS by the professional antigen presenting cells (APC) cells ofthe MHC class pathway. One example of this is a construct made with thepeptide transducing domain (PTD). In general, an immune response relieson native antigen processing and presentation. The tumor-associatedantigens can be expressed in bacteria, yeast or mammalian cells, howeverprotein antigens expressed in those systems likely will not maximallystimulate T cell responses (either CTL responses or Th1-biasedresponses) since the soluble exogenous proteins are processed mainly bythe MHC class II pathway. In fact, many anti-tumor vaccines rely on theinduction of CD8+ CTL, but this usually requires that the protein issynthesized within the cytosol of APC. Unfortunately, in general theplasma membranes of eukaryotic cells are impermeable to the majority ofproteins. It has recently been shown, however, that foreign proteinsfused with the protein-transducing domain (PTD) can penetrate the plasmamembrane, allowing the proteins to accumulate within the cells. Thisenhances the presentation of foreign peptides by the MHC class Imolecules of APCs to the antigen-specific CD8+ T cells.

Vaccination/Immunization

Vaccine formulations of the present invention comprise an immunogenicamount of a polynucleotide encoding a nonfunctional mutant BORISprotein, polypeptide or peptide, a nonfunctional mutant BORIS protein,polypeptide or peptide or a dendritic cell expressing a nonfunctionalmutant BORIS protein, polypeptide or peptide in combination with apharmaceutically acceptable carrier. Mimeotopes, which are polypeptidesof unrelated sequence but with a 3-dimensional structure correspondingto the nonfunctional mutant BORIS protein, polypeptide or peptide andthat immunologically function in an identical manner can be used.Mimeotopes, which are any biological molecule that is unrelated to BORISstructure, but has identical 3-d antigenic epitope/s and can berecognized by anti-BORIS T cells.

An “immunogenic amount” is an amount of the polypeptide encoding anonfunctional mutant BORIS protein, polypeptide or peptide,nonfunctional mutant BORIS protein, polypeptide or peptide or adendritic cell expressing a nonfunctional mutant BORIS protein,polypeptide or peptide sufficient to evoke an immune response in thesubject to which the vaccine is administered The amount administered isan amount that induces a desired immune response, and the desired degreeof protectionExemplary pharmaceutically acceptable carriers include, butare not limited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution.

The vaccine formulations of the present invention are suitable forpatients diagnosed with having at least one type of cancer including,but not limited to, breast, prostate, ovarian, gastric, liver,endometrial, glioma, colon, and esophageal cancer. The vaccineformulations of the present invention are also suitable for patientsknown to have a genetic susceptibility to cancer. In addition, thevaccine formulations of the present invention are suitable for thegeneral population at large, including those without cancer or without agenetic susceptibility to cancer, who wish to invoke protection againstcontracting at least one type of cancer that expresses the wildtypeBORIS protein, polypeptide or peptide.

Administration of the vaccine formulation may be carried out by anysuitable means, including parenteral injection (such as intraperitoneal,subcutaneous, or intramuscular injection), intradermal, intravenous,nasal, rectal, vaginal or to an airway surface. Topical application ofthe virus to an airway surface can be carried out by intranasaladministration (e.g. by use of dropper, swab, or inhaler which depositsa pharmaceutical formulation intranasally). Topical application of thevirus to an airway surface can also be carried out by inhalationadministration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the replicon as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. An “immunogenic amount” is an amount of the replicon particlessufficient to evoke an immune response in the subject to which thevaccine is administered.

When RNA or DNA is used as a vaccine, the RNA or DNA can be administereddirectly using techniques such as delivery on gold beads (gene gun),delivery by liposomes, or direct injection, among other methods known topeople in the art. Any one or more constructs or replicating RNA can beuse in any combination effective to elicit an immunogenic response in asubject. Generally, the nucleic acid vaccine administered may be in anamount that will induce a desired immune response, and the degree ofprotection desired. Precise amounts of the vaccine to be administeredmay depend on the judgment of the practitioner and may be peculiar toeach subject and antigen.

The vaccine may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of vaccination may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the immuneresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitableimmunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or otherschedules sufficient to elicit the desired immune responses expected toconfer protective immunity, or reduce disease symptoms, or reduceseverity of disease.

Human (hBORIS) can be isolated from human testis and manipulated in thesame manner. Likewise, BORIS can be isolated from the testis of anymammal or vertebrate and used similarly.

EXAMPLES

1. Generation of a Plasmid Encoding the ZF Deleted Form of the mBORISMolecule Under the hEF1-HTLV Promoter

An RT-PCR reaction is performed using poly-A RNA from mouse testis andthe following primers: MB1F 5′-CGTCACCATGGCTGCCGCTGAGGTCCCTG MB1R5′-AAGCTTCTGAAAGCTCTGAGGCTTTCCCTTGG MB2F5′-GGATCCGAGACGTTAGCCCCCAACAAGGACAGG MB2R5′-GAATTCTCACTTATCCATCATGTTAAAGATCATCTCGCAGG SpF5′-AGCTTGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGA TCGG SpR5′-GATCCCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCC ACCA

The PCR conditions are: 94° C. for 30 s, 60° C. for 30 s, 72° C. for 2min. Thirty (30) cycles are performed.

The PCR products are subcloned into the PCRII-TOPO cloning vector(Invitrogen). The C-terminal cDNA in PCRII-TOPO is restricted with BamHIenzyme and positive clones containing the insert are pooled andsubsequently restricted with HindIII enzyme. Spacer primers (SpF andSpR) are annealed to create overhanging sticky ends and ligated into theBamHI-HindIII restricted vector. The N-terminal encoded fragment isrestricted with HindIII and the inserts which are now separated from thevector are pooled and ligated into a HindIII digested constructcontaining the C-terminus and spacer. Clones with the proper orientationare then selected, sequenced (see, for example, the sequence for the ZFdeleted BORIS molecule below) and subcloned into the pORF plasmid undercontrol of the hEF1-HTLV promoter (Invivogen).

CHO cells are transfected with the resulting construct using standardmolecular techniques (Sambrook J, Fritsch E F and Maniatis T (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; incorporatedherein by reference)

The expression of the zinc finger deleted mBORIS construct is analyzedby Northern blots of mRNA isolated from transfected CHO cells usingstandard molecular technique (Sambrook et al., 1989).

2. Immunization of Mice with DNA Encoding the ZF Deleted Form of theBoris Molecule.

The plasmid encoding the ZF deleted mBORIS construct is isolated usingthe EndoFree Plasmid maxi kit (Qiagen). Purity of the plasmid DNA wasconfirmed by UV spectrophotometry (260 nm/280 nm absorbance ratio>1.7)and gel electrophoresis.

Gold beads are coated with DNA (1 μg/0.5 mg gold) and 5-7 weeks oldBalb/c mice are immunized using the Helios Gene Gun. Mice are boosted inthe same way, three times bi-weekly Ten days after the last boost, themice are bled and challenged with 1.0×10⁴ or 1.0×10⁵ 4T1 breast cancercells. The tumor size is measured everyday or every two-three singcalipers.

Protective Studies

Immunization of Mice with Plasmid Vaccine:

One example of a preventive anti-cancer vaccine approach is the use ofDNA encoding a deleted form of the mouse BORIS molecule that lacks thezinc finger domains and therefore the DNA binding property.

Purified plasmid is used to coat gold beads (2 μg plasmid/0.5 mg goldparticles) as was we described earlier (Ghochikyan et al.(2003) Eur JImmunol 33:3232-41). Immunizations of BALB/c mice with plasmid isperformed on shaved abdominal skin using the Helios gene gun(Bio-Rad,Hercules, Calif.) as described by Ross et al. (2000, Nat. Immunol1:127-131). Briefly, mice are bombarded 3 times with doses containing 2μg of DNA per 0.5 mg of˜1 μm gold beads (DeGussa-Huls Corp., RidefieldPark, N.J.) at a helium pressure setting of 400 psi. Mice are immunizedand boosted by the same method biweekly and challenged with twodifferent doses of 4T1 breast cancer cells (10⁵ or 10⁴) ten days afterthe last boost as described (Vasilevko et al., 2003). Different groupsof mice are immunized with plasmid encoding a modified BORIS moleculemixed with DNA encoding a certain molecular adjuvant(s) (see Table 1 fordetails). Such molecular adjuvants are known to increase cellular immuneresponses to the different antigens. TABLE 1 Mice were immunized fivetimes biweekly using a BORIS protective vaccine (pORF-mBORIS) mixed withpORF-mGMCSF (encoding mouse GMCSF), pORF-mIFNγ (encoding mouse IFNγ), orpORF-mIL12 + pIRES-mIL18 (two plasmids encoding mouse IL12 and IL18,accordingly). After the last boost, mice are challenged with 10⁵ or 10⁴4T1 mouse breast cancer cells that expressed the modified BORISmolecule. Challenge with Groups Immunogen Molecular Adjuvant 4T1 cells 1pBORIS pGM-CSF 10⁵ 2 pBORIS pIFNγ 10⁵ 3 pBORIS pIL12/IL18 10⁵ 4 Vector —10⁵ 5 — pIL12/IL18 10⁵ 6 pBORIS pIL12/IL18 10⁴ 7 Vector — 10⁴ 8 —pIL12/IL18 10⁴

Generation of Adenoviral Vector Encoding the ZF Deleted Form of MouseBORIS Molecule (Ads-BORIS) and Immunization of Mice

Ad5-BORIS recombinant adenovirus is prepared using AdEasy XL AdenoviralVector System from Stratagene. The shuttle vector is constructed bysubcloning the ZF-deleted mBORIS fragment into the plasmid pShuttle-CMV.For this purpose the BORIS fragment is synthesized by PCR usingpORF-mBORIS plasmid as a template and the following primers: SalI-MB-F5′-ACGCGTCGACATGGCTGCCGCTGAGGTCCCTGTCCCTTCTGGG NotI-MB-R5′-CGGCCGTCACTTATCCATCATGTTAAAGATCATCTCGCAGG

The PCR product is subcloned into the PCR4-TOPO cloning vector(Invitrogen). BORIS fragment is restricted using SalI and NotIrestriction endonucleases. The resulting product is purified on anagarose gel and subcloned using SalI-NotI cloning sites into thepShuttle-CMV vector.

The in vitro expression of ZF-deleted mBORIS is analyzed in CHO cells byimmunoblotting (see Figure below).

The shuttle vector carrying the deleted BORIS is linearized with PmeIand purified on an agarose gel. Electroporation competent cellsBJ-5183-Ad-1 are transformed with Pme-digested pShuttle-mBORIS plasmidto produce the recombinant Ad plasmid. AD-293 cells are transfected withselected recombinant Ad-BORIS DNA and primary viral stocks are prepared.The primary viral stock resulting (10⁷ pfu/ml) is amplified in AD-293cells and then purified on a CsCl gradient. The purified virus isdialyzed against PBS-5% sucrose and used for immunization of mice.

Balb/c mice immunized with pBORIS four times biweekly boosted once. i.m.with Ad5-BORIS (10⁹ PFU). Control animals injected with vectors areboosted with Ad5. Ten days after the last boost mice are challenged withtwo different doses of 4T1 breast cancer cells (10⁵ and 10⁴) asdescribed (Vasilevko et al., 2003).

Tumor Cell Lines

Mammary tumor cell lines provided by Dr. F. Miller (Karmanos CancerInstitute, Detroit, Mich.) are used. 4T1.2 cells are athioguanine-resistant variant derived from 410.4 cells (a mammary tumorcell line originally isolated from a single spontaneously arisingmammary tumor in a BALB/c fC3H mouse) without mutagen treatment. Thecells are cultured (37° C., 10% CO₂) in Dulbecco-modified Eagle'sessential medium (DMEM) containing low glucose and supplemented with 5%fetal bovine serum, 5% newborn calf serum, 2 mM glutamine, 100 units/mlpenicillin, 100 μg/ml streptomycin, 0.1 mM non-essential amino acids and1 mM sodium pyruvate (D10) (Life Technologies, Inc.).

Determination of Tumor Volumes

Tumor volumes are determined daily by two-dimensional measurement andcalculation using the formula L×(W²)/2, where L represents the lengthand W the width of the tumor. The experiments are terminated when themouse appears moribund or the tumor reaches approximately 1.5 cm³ forexperiments involving a challenge with 10⁴ cells and 2 cm³ forexperiments involving a challenge with 10⁵ 4T1 cells.

The time of appearance (latency period) is designated as the timeelapsed before a tumor with a volume in excess of 0.1 cm³ is present. Todetermine tumor growth rate, scatter plots are analyzed in the nearlinear periods of tumor growth.

Statistical Analysis

Results on the average times of appearance of tumor nodules (latencyperiod) and growth of tumor (tumor volume), as well as survival timesare examined using an analysis of variance (ANOVA) and Tukey multiplecomparisons post-test. Mean and standard deviation (SD) is calculatedusing GraphPad Prism 3.0 Software.

4. Immunology

B and T cell immune responses against BORIS are analyzed using twodifferent immunization protocols.

1. Preparation of Mouse BORIS Proteins and Immunization of Mice.

The ZF-deleted fragment of mouse BORIS is subcloned into the bacterialexpression vector pET24d(+) by using NcoI-XhoI cloning sites in framewith a C-terminal 6His tag. Both sites are introduced and a stop codonis removed during the PCR step of cloning. In addition, plasmidsencoding the deleted BORIS molecule fused with a Protein TransductionDomain (PTD) are constructed. The HIV-Tat protein transduction domain(Tat₄₇₋₅₇ YGRKKRRQRRR) is fused to the N-terminal end of the deletedBORIS via PCR and then cloned into the pET24d(+) vector NcoI-XhoIcloning sites. An E.coli BL21(DE3) strain transformed with the resultamtpET-mBORIS or pET-TATmBORIS plasmids, is grown in LB with kanamycin at28° C. until an A₆₀₀ 0.8 is reached. Protein synthesis is induced by theaddition of IPTG at a final concentration of 1 mM. The cells areharvested three to five hours later by centrifugation and used forprotein purification by affinity chromatography on a nickel-NTA(nitrilotriacetic acid) column (Qiagen).

In addition, the ZF-deleted fragment of BORIS fused with PTD issubcloned into the yeast expression vector pGAPZalpha in frame withsignal sequence into EcoRI-XbaI cloning sites. Both sites are introducedand the ATG initiation codon is removed during the PCR step of cloning.Pichia pastoris X33 strain is transformed by electroporation withpGAPZ-BORIS linearized with the AvrII restriction enzyme and positiveclones are selected on YPD media containing 100 μg/ml Zeocin. Forexpression analysis, selected positive clones are grown in YPD/Zeocinbroth and expression analyzed in supernatant at different time points byimmunoblotting.

2. Immunization of Mice with Dendritic Cells (DC).

Primary bone marrow DCs are obtained from mouse bone marrow precursorsas follows. Erythrocyte-depleted murine bone marrow cells harvested fromfemurs and tibias are plated in completed RPMI-10 media supplementedwith recombinant murine GM-CSF (100 U/ml). On day 3, nonadherentgranulocytes are gently removed and fresh media is added. Nonadherent DCare harvested at day 7 and purified by positive selection kit (Miltenyi)using CD11c Microbeads.

DC harvested at day 7 of culture and purified by positive selection areinfected with Ad5-BORIS by incubation at 10⁷ cells/ml in RPMI 1640 at amultiplicity of infection of 1000-2000. After 1 h, complete medium isadded to dilute the DC to a final concentration of 1×10⁶ to 2×10⁶cells/ml. Cells are harvested 24 h later, extensively washed in order todiscard any carryover of adenoviral particles, and used forimmunization. In addition, DC that are harvested at day 7 of culture andpurified by positive selection are incubated with 10 μg/ml ZF-deletedmBORIS protein at 37° C., 5% CO₂ for 24 hours, washed twice with PBS.Protein uptake by DC is analyzed in aliquots by Flow cytometry usinganti-mouse BORIS antibodies and appropriate secondary antibodies labeledwith FITC. Balb/c mice are immunized i.p. three times every three weekswith 1×10⁶ DC and T cell responses are analyzed 10 days after the lastboost in the cultures of splenocytes culture.

5. Results

Immunization results are presented in FIGS. 1-4.

DNA encoding a mutant form of the cancer-specific mouse BORIS antigenlacking DNA-binding function (deleted 11-Zinc Fingers) was constructedusing the mammalian expression vectors pORF (Invivogen) and the AdEasyXL Adenoviral Vector System (Stratagene). These vaccines have been usedas a prophylactic anti-cancer vaccine in a mouse breast cancer model. Inthis model we used BALB/c mice (H-2d haplotype) and the 4T1 nativemammary tumor cell line, which is a thioguanine-resistant variantderived from 410.4 cells without mutagen treatment. Importantly, thesemouse breast cancer cells are expressing the full mouse BORIS moleculeas we demonstrated by RT-PCR. Therefore this is an ideal model forexamination of ability of the BORIS molecule to be used as a protectivecancer vaccine.

Two different types of experiments were conducted. The first type ofexperiments included a group of mice that were vaccinated with pBORIS(plasmid encoding deleted mouse BORIS molecule) mixed with DNA encodingdifferent mouse cytokines (pGM-CSF; pIL12/IL18; pIFNγ) as molecularadjuvants. Mice were injected with vector (pORF) or pIL12/IL18 ascontrols. Mice were immunized and boosted using a gene gun technique andthen were challenged with 10⁴ or 10⁵ 4T1 cells.

The second type of experiment included a group of mice that werevaccinated with pBORIS and boosted with replication defective adenoviralvector (Ad5) that was modified to express the ZF deleted mouse BORISmolecule (Ad5-BORIS). A group of mice injected with vector and boostedwith Ad5 was used as a control. The animals were challenged with 10⁴ or10⁵ 4T1 cells and tumor appearance and growth were analyzed. We notethat it had previously been found that injection of as few as 10⁴ 4T1.2cells into the mammary glands of BALB/c mice resulted in local growth ofmammary tumors in 100% of challenged animals.

Vaccination with pBORIS plus pIL12/IL18 or pBORIS followed by Ad5-BORISresulted in protection of mice from challenge with 10⁴ unmodified 4T1tumor cells. Although 50% of the mice from the group immunized withpBORIS mixed with pIL12/IL18 generated small tumors (0.2-0.4 cm³), theyall survived by day 39. All experimental mice died approximate 10 daysearlier.

The results for mice vaccinated with Ad5-BORIS were more extreme. On day24, when mice in the control groups died from tumor growth, 100% of themice immunized with the Ad5-BORIS vaccine were not only alive, but didnot generate tumors at all. In fact they did not generate tumors atleast till day 33 after the challenge. These results indicate that theZF deleted BORIS vaccine effectively protected animals from a challengewith 10⁴ mammary tumor cells.

A second set of experiments was conducted using more stringentconditions and challenging mice with 10⁵ 4T1 tumor cells. Vaccinationwith the plasmid pBORIS plus pIFNγ or pIL12/IL18 significantly prolongedthe time of tumor growth to a volume of 2 cm³ and increased the survivalof the BALB/c mice. The vaccination also lowered the tumor growth ratein mice that were challenged with 10⁵ 4T1 tumor cells. A more profoundeffect was detected in mice vaccinated with pBORIS and boosted withAd5-BORIS before challenge with 10⁵ unmodified 4T1 cells. Here, on day23, when all mice in the control group had died from tumor growth, 80%of the mice immunized with Ad5-BORIS were alive and surviving animalshad significantly smaller sized tumors.

Separate groups of BALB/c mice were immunized with deleted mouse BORISprotein purified from E.coli system. Here, five mice were injectedsubcutaneously with protein (50 ug/mouse) mixed with Quil A Th1-typeconventional adjuvant (Sigma). After 4 immunizations all animals inducedsignificant titer of anti-BORIS antibodies. Another group of 5 mice weresimultaneously immunized i/p with isolated dendritic cells infected withAd5-BORIS. After 3 injections, the mice generated T cell responsesagainst mBORIS that were detected in vitro in the culture of splenocytesactivated with mBORIS protein. Therefore, immunization with BORISinduces B and T cell immune responses in mice and these immune responsesare protecting the animal from challenge.

6. Truncated BORIS Attached to the PTD as a Subunit Vaccine.

A PTD is attached to a nonfunctional truncated or mutant BORIS proteinand the fusion product generated in a yeast expression system. Genesencoding PTD and a nonfunctional mutant BORIS are subcloned into a yeastexpression vector such as pGAPZα. The expressed and secreted protein ispurified using standard molecular techniques. Mice are immunized withthe antigen formulated in two different conventional adjuvants andimmune responses as well as protection to the tumor antigen analyzed.

7. Virus-Like Particles Encoding a Nonfunctional Mutant BORIS as aSubunit Vaccine

A VLP-BORIS subunit vaccine based on Hepatitis B virus (HBV) coreantigen (HBcAg) is generated. This antigen self-assembles into VLPsafter expression in yeast cells. Foreign sequences can be inserted intoseveral regions of the HBcAg without disrupting the assembly process.Accordingly, a chimeric HBcAg-BORIS particle is generated that is usedfor immunization of mice.

8. Analysis in BALB/c and p53 KO Mice

Immune responses in BALB/c mice without challenge and in young p53knockout mice that are not developing tumors in that age are analyzed.Both humoral and cellular immune responses in mice immunized withdifferent BORIS vaccines are determined. Sera from immunized mice isanalyzed for detection of anti-BORIS antibody production during a 3month experimental period. CD4+ and CD8+ T cell proliferation andactivation of T regulatory cells before and after the challenge ofBALB/c mice is determined. Simultaneously, activation of NK cells thatcould directly kill mammary tumor cells is analyzed. Functional activityof BORIS-specific cytotoxic T lymphocytes (CTL) before and afterchallenge with mammary tumor 4T1 cells is demonstrated. P815 tumor cellsthat naturally express wildtype BORIS molecules are used as target cellsalong with 4T1 cells for detection of NK and CTL activity

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The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

Sequence Description: Nucleotide Sequence of ZF Deleted BORIS Moleculeatg gct gcc gct gag gtc cct gtc cct tct ggg tacttc acc cag atc aaa gag cag aag ttg aag cct ggagac cta gag gag gag aaa gag gag gac ggg gta caaaga gtg gaa gcc cag gag gga gtt gtc aag gag gtggag gcc gag aac agt tgc ctg ctt ctg gag gcc agggcc ccg gtg gag agc gac agg cgg atc ctg acc ctgcaa acg gtg cac ctg gag tcc cag gat gtg cac ctacag ggg ctg gga tgg ctg agc gtg cca cac tct gaggag ctt tca ggg acg gta cca gag gcg gaa ggc atactg cag ttg cca tcc gtg ctg tgg ctc gac cca gagccc cag ctc agc ctt cag cat tgc gtg acg gtc agcatc ccg gaa gag ctg tac cca cca gag gag ctg cagcgg ata cat ttt cac ctg ctg aga gag aat gtg ctaatg gcc gag gag aac cca gag tta aca cca gac ttggac gaa agc aca gcc ctg aaa aag ccc gaa gaa gatgaa aag gac cag ctc ccg ccc cag gga gag aca gacaag aga gaa gag agg ttg ctc ctt ctg gaa atg aaacca aaa gag gga aaa gac gac gaa att gtc ctg accatt tcc cat cta agc ctc gaa gaa cag caa gat ccacca gcg gcc aat cag aca agt gtg ccg gga gcc aaagcc gca aaa cca aaa cgg cgg agg cag acc aag ggaaag cct cag agc ttt cag aag ctt ggt gga ggc ggttca ggc gga ggt ggc tct ggc ggt ggc gga tcg ggatcc gag acg tta gcc ccc aac aag gac agg aga ccagtg aca agg aca cag gcc tcg gag gga gaa gca ggacac aag gaa ggg gag cct cag tgc cct ggg gag caggct ctg ggc cac caa gga gaa gca gcg ggg agc cagagc cca gac cac ggc ctt acc tgc gag atg atc ttt aac atg atg gat aag tga

Sequence Description: Wildtype Nucleotide Sequence of Mouse BORISCCATTTTGTGCACCTTGATCAAAGCCCATGTCTACTAGGCCCCAGCACCTCTGCACCCCATAAAGATTGCACGCTCTTTTTCCATCAGGGGTCGTCACCATGGCTGCCGCTGAGGTCCCTGTCCCTTCTGGGTACTTCACCCAGATCAAAGAGCAGAAGTTGAAGCCTGGAGACCTAGAGGAGGAGAAAGAGGAGGACGGGGTACAAAGAGTGGAAGCCCAGGAGGGAGTTGTCAAGGAGGTGGAGGCCGAGAACAGTTGCCTGCTTCTGGAGGCCAGGGCCCCGGTGGAGAGCGACAGGCGGATCCTGACCCTGCAAACGGTGCACCTGGAGTCCCAGGATGTGCACCTACAGGGGCTGGGATGGCTGAGCGTGCCACACTCTGAGGAGCTTTCAGGGACGGTACCAGAGGCGGAAGGCATACTGCAGTTGCCATCCGTGCTGTGGCTCGACCCAGAGCCCCAGCTCAGCCTTCAGCATTGCGTGACGGTCAGCATCCCGGAAGAGCTGTACCCACCAGAGGAGCTGCAGCGGATACATTTTCACCTGCTGAGAGAGAATGTGCTAATGGCCGAGGAGAACCCAGAGTTAACACCAGACTTGGACGAAAGCACAGCCCTGAAAAAGCCCGAAGAAGATGAAAAGGACCAGCTCCCGCCCCAGGGAGAGACAGACAAGAGAGAAGAGAGGTTGCTCCTTCTGGAAATGAAACCAAAAGAGGGAAAAGACGACGAAATTGTCCTGACCATTTCCCATCTAAGCCTCGAAGAACAGCAAGATCCACCAGCGGCCAATCAGACAAGTGTGCCGGGAGCCAAAGCCGCAAAACCAAAACGGCGGAGGCAGACCAAGGGAAAGCCTCAGAGCTTTCAGTGTGACACCTGCCCGTTCACTTCCTCCAAGCTCTCAACTTTCAATCGTCACATCAAAATTCACAGCAATGAGAGGCCACACCTGTGTCACCTGTGCCTGAAGGCCTTCCGGACTGTCACTCTTCTTAGGAACCATGTGAACACCCACACAGGAACCAGGCCCCACAAGTGCAGGGACTGCGACATGGCGTTTGTCACCAGCGGAGAACTCGTCCGGCACAGGCGTTACAAACACACTTATGAGAAGCCCTTCAAGTGCTCCCTGTGCAAGTACGCCAGCGTCGAGGCAAGCAAGATGAAGCGTCACATCCGCTCACACACGGGTGAGCGTCCCTTCCAGTGTTGCCAGTGTGCTTATGCCAGCAGGGACTCCTACAAGCTGAAGCGCCACATGAGGACACACTCAGGTGAGAAGCCGTATGAATGTCCCACCTGTCACGTCCGGTTCACCCAGAGCGGGACCATGAAAATCCATATAGCACAGAAGCACGGAGAGAATGTGCCCAAATACGAGTGTCCCCAGTGTGCCACCATCATCGCGAGGAAGAGCGACCTGCGTGTCCATCTGCGTAACCTGCACAGCCAGAGCCCGGAGGAGATGAAGTGCCGATACTGTCCCGCTGGCTTCCATGAGCGCTATGCCCTCATTCAGCACCAGAGGACCCACAAGAACGAGAAGAAGTTCAAGTGCAAGCAGTGCGATTACGCGTGCAAGCAGGAGCGATGCTTGAAGGCGCACATGCGCATGCACACAGGAGAGAAGCCCTTCTCCTGCCTGGCCTGCAACAAGCACTTCCGACAGAAGCAGCTACTGACCGTGCACCTGAGGAAGTACCATGACCCGAACTTCGTCCCCAATCTGCACCTGTGCCTCAAGTGTGATAAACGTTTCTCCCGCTGGAGTAACCTGCAGAGACACAGAAAGAAGTGTGACCCGGAGCATGAGACGTTAGCCCCCAACAAGGACAGGAGACCAGTGACAAGGACACAGGCCTCGGAGGGAGAAGCAGGACACAAGGAAGGGGAGCCTCAGTGCCCTGGGGAGCAGGCTCTGGGCCACCAAGGAGAAGCAGCGGGGAGCCAGAGCCCAGACCACGGCCTTACCTGCGAGATGATCTTTAACATGATGGATAAGTGATGGATAAGTGAGCAGTCGTGCCTCTCCGTGCAGTGGCCTCTGGGGGAAGAAACCAGTTAGAAATAAGTTCCCAGACACAGCACAGTGTTCTCAGAGTTTGAGATAGTGTGTAGAAATGTTTGAGAGAAGGGGAAAAAAACCCTGCAGCTATTTCCAAAGACTTGAGTCAGAGCTCGAAGTGAAGGTGCACATATCTGGGCCCTAGCAGGTGCCCAGAATGAGTCAGGGACAGATTCTAGGTGATACTTATGTCCACGGGGGCTCAGACCAGTTAACGCCTTGGTGGTCAGAGCAGAAAATTTTTTGAGTTGTTGTACCCACCCTCAA

Sequence Description: Wildtype Nucleotide Sequence of Human BORIS 1ggcaccagac gcggtgcacg aggcagagcc acaagccaaa gacggagtgg gccgagcatt 61ccggccacgc cttccgcggc caagtcatta tggcagccac tgagatctct gtcctttctg 121agcaattcac caagatcaaa gaactcgagt tgatgccgga aaaaggcctg aaggaggagg 181aaaaagacgg agtgtgcaga gagaaagacc atcggagccc tagtgagttg gaggccgagc 241gtacctctgg ggccttccag gacagcgtcc tggaggaaga agtggagctg gtgctggccc 301cctcggagga gagcgagaag tacatcctga ccctgcagac ggtgcacttc acttctgaag 361ctgtggagtt gcaggatatg agcttgctga gcatacagca gcaagaaggg gtgcaggtgg 421tggtgcaaca gcctggccct gggttgctgt ggcttgagga agggccccgg cagagcctgc 481agcagtgtgt ggccattagt atccagcaag agctgtactc cccgcaagag atggaggtgt 541tgcagttcca cgctctagag gagaatgtga tggtggccag tgaagacagt aagttagcgg 601tgagcctggc tgaaactgct ggactgatca agctcgagga agagcaggag aagaaccagt 661tattggctga aagaacaaag gagcagctct tttttgtgga aacaatgtca ggagatgaaa 721gaagtgacga aattgttctc acagtttcaa attcaaatgt ggaagaacaa gaggatcaac 781ctacagctgg tcaagcagat gctgaaaagg ccaaatctac aaaaaatcaa agaaagacaa 841agggagcaaa aggaaccttc cactgtgatg tctgcatgtt cacctcttct agaatgtcaa 901gttttaatcg tcatatgaaa actcacacca gtgagaagcc tcacctgtgt cacctctgcc 961tgaaaacctt ccgtacggtc actctgctgc ggaaccatgt taacacccac acaggaacca 1021ggccctacaa gtgtaacgac tgcaacatgg catttgtcac cagtggagaa ctcgtccgac 1081acaggcgcta taaacatact catgagaaac cctttaaatg ttccatgtgc aagtatgcca 1141gtgtggaggc aagtaaattg aagcgccatg tccgatccca cactggggag cgcccctttc 1201agtgttgcca gtgcagctat gccagcagag atacctacaa gctgaaacgc cacatgagaa 1261cgcactcagg tgagaagcct tacgaatgcc acatctgcca cacccgcttc acccagagcg 1321ggaccatgaa aatacatatt ctgcagaaac acggcgaaaa tgtccccaaa taccagtgtc 1381cccattgtgc caccatcatt gcacggaaaa gcgacctacg tgtgcatatg cgcaacttgc 1441atgcttacag cgctgcagag ctgaaatgcc gctactgttc tgctgtcttc catgaacgct 1501atgccctcat tcagcaccag aaaactcata agaatgagaa gaggttcaag tgcaaacact 1561gcagttatgc ctgcaagcag gaacgtcata tgaccgctca cattcgtacc cacactggag 1621agaaaccatt cacctgcctt tcttgcaata aatgtttccg acagaagcaa cttctaaacg 1681ctcacttcag gaaataccac gatgcaaatt tcatcccgac tgtttacaaa tgctccaagt 1741gtggcaaagg cttttcccgc tggattaacc tgcacagaca ttcggagaag tgtggatcag 1801gggaagcaaa gtcggctgct tcaggaaagg gaagaagaac aagaaagagg aagcagacca 1861tcctgaagga agccacaaag ggtcagaagg aagctgcgaa gggatggaag gaagccgcga 1921acggagacga agctgctgct gaggaggctt ccaccacgaa gggagaacag ttcccaggag 1981agatgtttcc tgtcgcctgc agagaaacca cagccagagt caaagaggaa gtggatgaag 2041gcgtgacctg tgaaatgctc ctcaacacga tggataagtg agagggattc gggttgcgtg 2101ttcactgccc ccaattccta aagcaagtta gaagttttta gcatttaagg tgtgaaatgc 2161tcctcaacac gatggataag tgagagagag tcaggttgca tgttcactgc ccctaattcc 2221taaagcaagt tagaaatttt tagcattttc tttgaaacaa ttaagttcat gacaatggat 2281gacacaagtt tgaggtagtg tctagaattg ttctcctgtt tgtagctgga tatttcaaag 2341aaacattgca ggtattttat aaaagtttta aaccttgaat gagagggtaa cacctcaaac 2401ctatggattc attcacttga tattggcaag gtggcccaca atgagtgagt agtgattttt 2461ggatatttca aaatagtcta gaccagctag tgcttccaca gtcaaagctg gacattttta 2521tgttgcatta tatacaccca tgatatttct aataatatat ggttttaaac attaaagaca 2581aatgttttta tacaaatgaa ttttctacaa aatttaaagc taccataatg cttttaatta 2641gttctaaatt caaccaaaaa atgttttact cttataaaaa ggaaaactga gtaggaaatg 2701aaatactaga ttagactaga aaataaggaa taaatcgatt ttactttggt ataggagcaa 2761ggttcacctt tagatttttg tattctcttt taattatgct ccttggcagg tatgaaattg 2821ccctggttac attccattat tgcttattag tatttcactc cataaccctt ttttctgcta 2881aaactactct ttttatattt gtaaaataat tggcagagtg agaagaaaca taaaatcaga 2941taaggcaaat gtgtacctgt aaggaatttg tactttttca taatgcccag tgattagtga 3001gtatttccct tttgccagtt gacaagattt ttccaccctc gagcagcgtg agagatgcct 3061ctttaacact tgaaattcat ttctatctgg atacagaggc agatttttct tcattgctta 3121gttgagcagt ttgttttgct gccaacctgt ctccacccct gtatttcaag atcattgata 3181agccctaaat tcaaattctt aagatatgga ccttttattg aaaatatcac aagttcagaa 3241tccctataca atgtgaatat gtggaaataa tttcccagca ggaagagcat tatattctct 3301ttgtaccagc aaattaattt aactcaactc acatgagatt taaattctgt gggctgtagt 3361atgccatcat tgtgactgaa tttgtgcaat ggtttcttaa tttttttact gttatttaaa 3421gatgttttac ataattcaat aaaatgaaat gacttaaaat tgcaaaaaaa aaaaaaaaaa 3481aaaaaaaaaa aaaaaaaaaa

Sequence Description: Amino Acid Sequence of Human BORISMAATEISVLSEQFTKIKELELMPEKGLKEEEKDGVCREKDHRSPSELEAERTSGAFQDSVLEEEVELVLAPSEESEKYILTLQTVHFTSEAVELQDMSLLSIQQQEGVQVVVQQPGPGLLWLEEGPRQSLQQCVAISIQQELYSPQEMEVLQFHALEENVMVASEDSKLAVSLAETAGLIKLEEEQEKNQLLAERTKEQLFFVETMSGDERSDEIVLTVSNSNVEEQEDQPTAGQADAEKAKSTKNQRKTKGAKGTFHCDVCMFTSSRMSSFNRHMKTHTSEKPHLCHLCLKTFRTVTLLRNHVNTHTGTRPYKCNDCNMAFVTSGELVRHRRYKHTHEKPFKCSMCKYASVEASKLKRHVRSHTGERPFQCCQCSYASRDTYKLKRHMRTHSGEKPYECHICHTRFTQSGTMKIHILQKHGENVPKYQCPHCATILARKSDLRVHMRNLHAYSAAELKCRYCSAVFHERYALIQHQKTHKNEKRFKCKHCSYACKQERHMTAHIRTHTGEKPFTCLSCNKCFRQKQLLNAHFRKYHDANFIPTVYKCSKCGKGFSRWINLHRHSEKCGSGEAKSAASGKGRRTRKRKQTILKEATKGQKEAAKGWKEAANGDEAAAEEASTTKGEQFPGEMFPVACRETTARVKEEVDE GVTCEMLLNTMDK

Sequence Description: Amino Acid Sequence of Mouse BORISMAAAEVPVPSGYFTQIKEQKLKPGDLEEEKEEDGVQRVEAQEGVVKEVEAENSCLLLEAR 60APVESDRRILTLQTVHLESQDVHLQGLGWLSVPHSEELSGTVPEAEGILQLPSVLWLDPE 120PQLSLQHCVTVSIPEELYPPEELQRIHFHLLRENVLMAEENPELTPDLDESTALKKPEED 180EKDQLPPQGETDKREERLLLLEMKPKEGKDDEIVLTISHLSLEEQQDPPAANQTSVPGAK 240AAKPKRRRQTKGKPQSFQCDTCPFTSSKLSTFNRHIKIHSNERPHLCHLCLKAFRTVTLL 300RNHVNTHTGTRPHKCRDCDMAFVTSGELVRHRRYKHTYEKPFKCSLCKYASVEASKMKRH 360IRSHTGERPFQCCQCAYASRDSYKLKRHMRTHSGEKPYECPTCHVRFTQSGTMKIHIAQK 420HGENVPKYECPHCATILARKSDLRVHLRNLHSQSPEEMKCRYCPAGFHERYALIQHQRTH 480KNEKKFKCKQCDYACKQERCLKAHMRNHTGEKPFSCLACNKHFRQKQLLTVHLRKYHDPN 540FVPNLHLCLKCDKRFSRWSNLQRHRKKCDPFHFTLAPNKDRRPVTRTQASEGEAGHKEGE 600PQCPGEQALGHQGEAAGSQSPDHGLTCEMIFNMMDK

Sequence Description: Amino Acid Sequence of ZF Deleted Mouse BORISMAAAEVPVPSGYFTQIKEQKLKPGDLEEEKEEDGVQRVEAQEGVVKEVEAENSCLLLEARAPVBSDRRILTLQTVHLESQDVHLQGLGWLSVPHSEELSGTVPEAEGILQLPSVLWLDPEPQLSLQHCVTVSIPEELYPPEELQRIHFHLLRENVLMAEENPELTPDLDESTALKKPEEDEKDQLPPQGETDKREERLLLLEMKPKEGKDDEIVLTISHISLEEQQDPPAANQTSVPGAKAAKPKRRRQTKGKPQSFQKLGGGGSGGGGSGGGGSGSETLAPNKDRRPVTRTQASEGEAGHKEGEPQCPGEQALGHQGEAAGSQSPDHGLTCEMIFNMMDK

1. An immunogenic composition comprising a member selected from thegroup consisting of a polynucleotide encoding a nonfunctional mutantform of the Brother of Regulator of Imprinted Sites (BORIS) protein,polypeptide or peptide, a nonfunctional mutant BORIS protein,polypeptide or peptide, a dendritic cell expressing a nonfunctionalmutant BORIS peptide, polypeptide or protein and any molecule thatmimics a nonfunctional mutant BORIS molecule.
 2. The compositionaccording to claim 1, wherein said nonfunctional mutant BORIS protein,polypeptide or peptide lacks the DNA-binding property.
 3. Thecomposition according to claim 1, wherein said nonfunctional BORISprotein, polypeptide or peptide lacks zinc finger domain.
 4. Thecomposition according to claim 1, wherein said protein, polypeptide orpeptide is attached to a pharmaceutically accepted carrier.
 5. Thecomposition according to claim 1, wherein said BORIS protein,polypeptide or peptide is attached to a protein transducing domain. 6.The composition according to claim 1, wherein said mutant BORIS protein,polypeptide or peptide is attached to a peptide that modifies BORIS andretains or enhances said BORIS protein, polypeptide or peptideantigenicity.
 7. The composition according to claim 1, wherein saiddendritic cell expressing said nonfunctional mutant BORIS protein,polypeptide or peptide is transfected with DNA encoding a mutant BORISprotein, polypeptide or peptide.
 8. The composition according to claim1, wherein said dendritic cell expressing said nonfunctional mutantBORIS protein, polypeptide or peptide is infected with the viral vectorencoding a nonfunctional mutant BORIS protein, polypeptide or peptide.9. The composition according to claim 1, wherein said dendritic cellexpressing said mutant BORIS protein, polypeptide or peptide is loadedwith BORIS protein, polypeptide or peptide or any modified protein,polypeptide or peptide form of BORIS.
 10. The composition according toclaim 1, further comprising an adjuvant.
 11. The composition accordingto claim 10, wherein said adjuvant is mixed or fused to said DNAencoding a nonfunctional mutant BORIS protein, polypeptide or peptide,or mutant BORIS protein, polypeptide or peptide.
 12. The compositionaccording to claim 10, wherein said adjuvant is selected from the groupconsisting of a cytokine, a chemokine and a costimulatory molecule. 13.A vector comprising an immunogenic composition according to claim
 1. 14.The vector of claim 13, wherein said vector allows expression inbacterial, mammalian, yeast or viral systems.
 15. A host celltransformed with the composition according to claim
 1. 16. The host cellaccording to claim 15, wherein said cell is a bacterial cell, mammaliancell or a yeast cell.
 17. An antibody comprising a binding site thatrecognizes an epitope from a nonfunctional mutant BORIS protein,polypeptide or peptide.
 18. A vaccine against cancer comprising amolecule selected from the group consisting of a polynucleotide encodinga nonfunctional mutant form of the BORIS protein, polypeptide orpeptide, a nonfunctional mutant BORIS protein, polypeptide or peptide,dendritic cells expressing a nonfunctional mutant BORIS peptide,polypeptide or protein and any molecules that mimics a nonfunctionalmutant BORIS molecule.
 19. The vaccine according to claim 18 whereinsaid nonfunctional mutant BORIS lacks at least one zinc-finger domain.20. The vaccine according to claim 18, wherein said nonfunctional mutantBORIS lacks the DNA binding capability or any other native BORISfunction.
 21. The vaccine according to claim 18, further comprising anadjuvant.
 22. The vaccine according to claim 1, further comprising anpharmaceutically acceptable carrier.
 23. A method of immunizing amammalian subject which comprises administering to a patient in needthereof an effective amount of a member of the group consisting of apolynucleotide encoding a nonfunctional mutant form of BORIS protein,polypeptide or peptide, a nonfunctional mutant BORIS protein,polypeptide or peptide, dendritic cells expressing a nonfunctionalmutant BORIS molecule and any molecules that mimics a nonfunctionalmutant BORIS molecule.
 24. The method of claim 23, wherein saidpolynucleotide encoding a nonfunctional mutant BORIS protein,polypeptide or peptide, a nonfunctional mutant BORIS protein,polypeptide or peptide, dendritic cells expressing a nonfunctionalmutant BORIS molecule or any molecules that mimics a nonfunctionalmutant BORIS molecule is mixed or fused with a molecular adjuvant. 25.The method of claim 23, wherein said molecular adjuvant is a moleculethat increases cellular immune response and/or antibody responses. 26.The method of claim 1, wherein said molecular adjuvant is selected fromthe group consisting of any cytokine, chemokine, costimulatory molecule.27. The method of claim 1, wherein said polynucleotide encoding anonfunctional mutant BORIS protein, polypeptide or peptide, anonfunctional mutant BORIS protein, polypeptide or peptide, dendriticcells expressing a nonfunctional mutant BORIS molecule or any moleculesthat mimics a nonfunctional mutant BORIS molecule is mixed with anyconventional or molecular adjuvant.
 28. The method of claim 1, whereinsaid mutant BORIS protein, polypeptide or peptide is attached topharmaceutically accepted carrier.
 29. The method of claim 1, whereinsaid mutant BORIS protein, polypeptide or peptide is attached to apeptide that modifies BORIS and retains and/or enhances its antigenicproperty.
 30. The method of claim 1, wherein said mutant BORIS protein,polypeptide or peptide further comprises a protein transducing domain(PTD).
 31. The method of claim 1, wherein said dendritic cellsexpressing BORIS are transfected with DNA encoding a mutant BORISmolecule.
 32. The method of claim 1, wherein said dendritic cellsexpressing BORIS are infected with viral vector encoding a nonfunctionalmutant BORIS molecule.
 33. The method of claim 1, wherein said dendriticcells expressing BORIS are loaded with BORIS protein, polypeptide, orpeptide, or any modified protein form of BORIS.
 34. The method of claim1, wherein said administration occurs via an intramuscular,subcutaneous, intradermal, intravenous, nasal, rectal, vaginal orperitoneal
 35. The method according to claim 1, wherein said patient hasmore than one type of cancer.
 36. The method of claim 1, wherein saidpatient has cancer.
 37. The method according to claim 1, wherein saidpatient does not have cancer.
 38. The method according to claim 1,wherein said patient has no cancer, but has a genetic susceptibility tocancer.
 39. The method according to claim 1, wherein said normal patienthas no detectable cancer, but desires to protect himself from possiblegeneration of malignancy.
 40. The method according to claim 1, whereinsaid immunization results from mounting a cellular immune responsecomprising T cells that recognize an epitope from a nonfunctional mutantBORIS peptide, polypeptide, protein or mimeotope.
 41. A dendritic cellexpressing a nonfunctional mutant BORIS peptide, polypeptide, protein ormimeotope.
 42. A dendritic cell loaded with a nonfunctional mutant BORISpeptide, polypeptide, protein or mimeotope.
 43. A dendritic cellexpressing a nonfunctional mutant BORIS protein, polypeptide or peptideinfected with the viral vector encoding a nonfunctional mutant BORISprotein, polypeptide or peptide.
 44. Use of an immunogenic compositionfor the preparation of a vaccine against cancer wherein said immunogeniccomposition comprises a member selected from the group consisting of apolynucleotide encoding a nonfunctional mutant BORIS protein,polypeptide or peptide, a nonfunctional mutant BORIS protein,polypeptide or peptide, dendritic cells expressing a nonfunctionalmutant BORIS molecule or any molecule that mimics a nonfunctional mutantBORIS molecule.